Bioengineering approaches to map mechanotransduction in the living cell Abstract Mechanical forces strongly influence the growth and form of virtually every tissue and organ in our bodies. Yet little is known about the mechanism by which individual cells sense these mechanical signals and transduce them into changes in intracellular biochemistry and gene expression - a process known as mechanotransduction. We find that, surprisingly, a local surface stress is concentrated in the cytoplasm and propagated rapidly along the cytoskeleton to distant sites to activate specific enzymes, representing drastic departures from existing prevailing models of mechanotransduction. In this revised renewal application we propose three aims. Aim 1 is to dissect the molecular differences between growth factor induced and stress-induced Src and Rac activation. Aim 2 is to test the hypothesis that physical interactions of nuclear proteins coilin-SMN can be directly altered by a local stress at the cell surface. Aim 3 is to elucidate mechanisms of mechanical signaling mediated spreading and differentiation in embryonic stem cells. The proposed bioengineering research combines mechanical quantification of the living cell with biochemical and biological measurements. The experimental approach is to measure with high spatial and temporal resolution the cytoplasmic and subnuclear structural deformation and simultaneously quantify biochemical activities, protein dynamics, and gene expressions in a living cell. The current project may have implications in elucidating specific loci and protein complexes of mechanotransduction at sites deep in the cytoplasm and the nucleus that are responsible for regulation of gene expression and differentiation. A growing body of evidence demonstrates that abnormal mechanical forces may contribute to the development of various diseases, such as atherosclerosis, asthma, progeria, and cancer progression, by altering form and function of living cells. The present study may provide a unique way to identify potential structural and molecular targets of mechanotransduction for therapeutic intervention in the future.